Process for the treatment of tobacco



United States Patent 3 256,889 PROCESS FOR THE TREATMENT OF TOBACCORoger L. de la Burd, 1617 Pope Ave., Richmond, Va., and Frank H.Crayton, 6343 Glyndon Lane, Richmond 25, Va. No Drawing. Filed Nov. 30,1962, Ser. No. 241,124

g 2 Claims. (Cl. 131-141) This invention relates to a process for thetreatment of tobacco. Moreparticularly, the invention [relates to aprocess for improving the smoking characteristics of tobacco.

It has long been a problem in the art that freshly harvested tobaccogenerally requires several years, involving a number of differenttreatments, before it is ready to be used in smoking products. Forexample, the harvested tobacco is generally dried for several months inorder to cure it and the cured tobacco thereafter undergoes a number ofsweating, redrying, resweating and aging operations which extend over aperiod of two to three years.

Numerous attempts have been made to shorten the period of time necessaryto convert freshly harvested tobacco to a smoking product which hasdesirable flavor and smoking qualities. For example, bacteria have beenadded to tobacco and catalysts have been incorporated in tobacco inorder to accelerate the fermentation process. However, none of theseattempts has been found to be completely satisfactory.

It is an object of the present invention to provide an improved processfor treating tobacco to improve the smoking characteristics thereof.

It is another object of the present invention to provide a process forup-grading tobacco to a satisfactory level of flavor and smokingqualities in a relatively short period of time.

It is also an object of the present invention to eliminate thedisadvantageous changes which occur in the moisture retention propertiesof tobacco which has been treated by the conventional aging processes.

Additional objects of the present invention will be apparent from thefollowing description.

I have discovered that the smoking characteristics of tobacco aregreatly improved by the addition of a peptidic enzyme to the tobacco.

In accordance with the present invention, a peptidic enzyme is added totobacco in an amount sufiicient to provide a tobacco compositioncontaining from about 0.02 to about 0.15 percent, and preferably fromabout 0.05 to about 0.12 percent, by weight of added peptidic enzyme.This corresponds to adding from about 0.02 to'about 0.15 part by weight,and preferably from about 0.05 to about 0.12 part by weight, of peptidicenzyme per 100 parts of tobacco.

The enzyme can be applied in any suitable manner. However, it isgenerally applied to the tobacco in the form of a liquid solution,suspension or emulsion by spraying, dipping, brushing or other means.

The enzyme is preferably added to the tobacco as a 0.5 to 1.5% aqueoussolution, with sufficient solution being added to bring the tobacco to amoisture content of 15 to 45% and is most preferably added to thetobacco as a 0.5 to 1.5 solution, with suflicient solution to bring thetobacco to a moisture content of 15 to 25% by weight.

The preferred method of adding the enzyme is to spray a solution of iton tobacco leaves, since by this method the moisture content of thesprayed leaves can readily be controlled. The pH of the enzyme solutioncan be adjusted to a value between about 5.2 and 8.5 by the addition ofsuitable agents, such as sodium hydroxide, ammonium hydroxide, diluteacetic and sulfuric acid, and the like but is preferably adjusted to avalue of from about 6.5 to 7.6, in order to best facilitate the actionof the enzyme.

The moisture content of the treated leaves is important and should befrom about 15% to about after the addition of enzyme. At least 15%moisture is necessary in order to produce a product having sufficientmildness. The upper limit of 40% is necessary, since raising themoisture content of the leaves above the level irreversibly decreasessome of the improved smoking characteristics which would otherwise beobtained by the present enzyme treatment of the leaves.

The addition of the enzyme to the tobacco can be conducted attemperatures of from 60 to 140 F. but is preferably conducted at roomtemperature.

The peptidic enzymes .of the invention include mono-, di-, andtri-polypeptidases, carboxy-peptidase, endopeptidases, aminoacylases,aminopeptidases, dehydropeptidases, and dipeptidases which havespecificity for amino acids, amino acid amides, diand tripeptides,polypeptides, and dehydropeptides. The best source of such enzymes ishog kidney although they can be extracted from intestinal mucosa, liver,and similar animal tissues. These enzymes may be employed either aloneor in combination.

In accordance with one embodiment of the present invention, the peptidicenzymes are added to the tobacco with proteolytic enzymes in the ratioof from 1:10 to 1:1, respectively. The proteolytic enzymes which can beemployed can be of the same type employed in my copending applicationSerial No. 241,123, filed of even date herewith. When these two enzymesare used in combination the amount employed should be such that thefirst product contains a total of from 0.28 to 0.56% by weight of addedenzymes in the ratios given above. Apart from this requirement, theaddition of the two types of enzymes will be the same and the remainderof the tobacco treatment can be the same as if the peptidic enzyme wereemployed by itself.

The enzyme should be evenly distributed throughout the tobacco leaves.It can then be placed in suitable containers, such as polyethylene bags,which will prevent evaporation of moisture in the mixture.

The resulting enzyme-tobacco mixtures can then be placed in anatmosphere, for example a humidity cabinet, maintained at 60 to 140 F.and 45 to 85% relative humidity for a period of from 1 to 200 hours.

The tobacco parts can then be removed from the above atmosphere and canbe placed in an environment maintained at a temperature of from 60 to 80F. and 60 to relative humidity, in order to permit the moisture contentof the leaves to reach a value of from about 11 to about 14%, preferablyabout 12%, by weight.

Tobaccos which can be treated in accordance with the present inventioncan be of any quality or origin. For example, the tobacco can be lowgrade or high grade domestic tobacco, for example bur-ley, Maryland,bright or Virginia tobaccos ormixtures of these. The tobacco samelow-grade bright tobacco. in the cigarette fillers.

can also be, for example, an imported aromatic or oriental tobacco ormixtures of these, including mixtures with EXAMPLE 1 Twenty-four .poundsof the full leaves of an unaged low-grade bright tobacco (RDZ) wereconditioned to moisture in a humidity cabinet and were sprayed at roomtemperature in separate two-pound batches with 0.2, 0.4, 0.6, 0.8, 1.0,1.2, 1.4, 2.0, 3.0, 4.0, 5.0 and 6.0% (by weight) aqueous solutions ofpeptidase (obtained from Nutritional Biochemicals Corporation) andcomprising a mixture of peptidic enzymes.

In each case, sufficient solution was employed to bring the moisturecontent of the leaves to -22% (by weight). Each batch of leaves sotreated was mixed thoroughly and packed loosely in polyethylene bags.The bags were closed to prevent evaporation of the moisture and werethen placed in humidity cabinets maintained at a temperature of 100 F.and at 70% relative humidity for 60 hours.

Following this treatment, the leaves were removed from the polyethylenebags and placed loosely on wire mats in a room maintained at atemperature of 75 C. and at a relative humidity of 60% until themoisture in the leaf had a value of about 12% by weight, which requiredabout 24 to 48 hours.

In each case, visual examination of the leaves showed no change in theappearance of the tobacco.

The treated tobacco leaves which had been sprayed with a 1.0% aqueoussolution of peptidase and untreated leaves of the same tobacco were eachseparately immersed for 15 minutes in a 0.1 N solution of sodiumhydroxide. The resulting solutions were dialyzed in a Visking dialysistube against distilled water. The liquids which remained in the dialysisbag were precipitated with ammonium chloride and the precipitates wereweighed. Since the -membrane of the dialysis tube did not allow thepassage of substances having a molecular weight higher than 10,000, thereduction of substances below the 10,000 molecular weight could becalculated from the treated tobacco and the untreated control. Areduction of approximately 40% was calculated, showing that the actionof the peptidase on the tobacco had produced a structural change withinthe leaf by reducing the molecular weight of the polypeptides in theleaf. The peptidase had cleaved the longchain peptides into lowermolecular weight units. The tobacco leaves which had been treated asabove with a 1.0% solution of peptidase and its control sample were alsoanalyzed for their free amino acid content. The tobacco waswater-extracted and the extract was examined on an automatic amino acidanalyzer; This analysis showed that the treatment with peptidaseincreased the free amino acid content by approximately 27%.

Cigarettes were made from each batch of the enzymetreated tobacco andfrom untreated unaged leaves of the No additives were used Subjectiveevaluations of the cigarettes by a smoking panel of 59 members showedthat the cigarettes from the enzyme-treatedv tobacco were preferred overcigarettes from the untreated tobacco. The panel found that the smokefrom the treated tobacco was much more mild than that from the controland that it had a note of sweetness, a fuller body, and a more desirableflavor than that from the control cigarette. Several of the smokersobserved that the desirable flavor increased in intensity during thesmoking of the cigarette.

Data on the subjective evaluation showed that there was an optimumconcentration level for the enzyme above and below which the developmentof flavor and aroma was not complete. This trend is shown by the datapresented below in Table I.

It can be seen from Table I that the best results were obtained when thepeptidase was applied at 0.6 to 1.4% concentration. Smoke from tobaccostreated with concentrations exceeding 3% was more harsh and had agreater bitey-ness than did the smoke from the control.

Table I TEST PANEL EVALUATIONS OF UNAGED LOW-GRADE BRIGHT TOBACCOCONTAINING PEPTIDASE 1 A scale of 5 to +5 was employed, 5, being thepoorest rating, 0 being the characteristics of the control, and +5 beingthe best rating.

Visual comparisons of the materials tested showed that the ash from thetreated cigarettes compared well with that from the untreated and thatthe burning rate and filling power of the tobacco had been improved bythe treatment.

EXAMPLE 2 Twenty-four pounds of the full leaves of a good grade brighttobacco (E7) were treated as described in Example 1 and sprayed at roomtemperature in separate two-pound batches with 0.2, 0.4, 0.6, 0.8, 1.0,1.2, 1.4, 2.0, 3.0, 4.0, 5.0, and 6.0% (by weight) aqueous solutions ofthe same type peptidase and in the same manner as employed in Example 1.

The leaves were further treated in the manner described in Example 1 andwere brought to a moisture content of about 12% by weight.

In each case, visual examination of the leaves showed no change in theappearance of the tobacco.

By using the methods described in Example 1, it was determined that themolecular weight of the polypeptides in the tobacco had been reducedabout 40%, showing that the peptidase had cleaved the long-chainpeptides into lower molecular weight units. Analyses of the free aminoacid content of the leaves, as described in Example 1, showed that thetreatment with peptidase had increased the free amino acid content byapproximately 30%.

Cigarettes were made from each batch of the enzymetreated tobacco andfrom untreated unaged leaves of the same good-grade bright tobacco. Noadditives were used in the cigarette filters.

Subjective evaluation of the cigarettes by a 59-member smoking panelshowed that the cigarettes from the enzymetreated tobacco were preferredover cigarettes from the untreated tobacco at a -1 probability level.The smokers found that the smoke from the treated tobacco was much moremild than that from the control, and that it had a fuller body, a noteof sweetness, and a more desirable aroma and flavor. Data on thesubjective evaluation showed a trend which indicated that the optimumresults were obtained when the peptidase was applied at a 0.4 to 1.4concentration. This trend can be seen from the data in Table II.

TEST PANEL EVALUATIONS OF GOOD GRADE B RIGHT TOBACCO CONTAININGPEPTIDASE Average Rating 1 From 59 Panelists Percent ConcentrationHarshness Aroma Preference 0.2 0. 0. 00 +0. 0.4 +0. 62 0. 00 +0. 050.6." 05 +0.12 +0.05 0.8.-. +1. 62 +1.12 +2. 62 1.0-.. +2. 66 +3. 25 +4.37 1.2. +1. 37 +2. 00 +2. 00 1 4 0. 00 +0. 33 +0.50 2 0. 62 0. 00 0. 333.0 2. 00 -l. 00 1.l2 4.0 2. 00 -1. 12 -1. 12 5.0 2. 00 l. 62 1. 266.0 1. 67 -2. 25 2. 00

1 Same scale as in Table I.

EXAMPLE 3 Five pounds of a good quality bright tobacco (RDA) of the cropyear 1959 was sprayed with a 1% aqueous solution of the peptidasedescribed in Example 1, in an amount suflicient to bring the moisturelevel of the leaves to 19%. The leaves were then treated as in Example1, and equilibrated to 60% RH. and 75 F. to bring the moisture contentto 12%.

Visual examination showed that the appearance of the tobacco wasunchanged.

Cigarettes were prepared in the conventional manner from the treatedtobacco and from naturally aged and unaged leaves of the samegood-quality stock. These cigarettes were smoked and evaluated in threedifferent smoking panel tests. The subjective evaluation by one of thetests with 31 panel members showed that the cigarettes from the treatedtobacco showed a slight trend toward an improvement in flavor over thecigarettesprepared from untreated naturally-aged tobacco. A trend inpreference for the cigarette from the treated tobacco was also ob- Insubjective tests by a 39-member smoking panel, the enzyme-treatedtobacco was judged by statistical evaluation to have lower mouthharshness and over all better smoking characteristics.

EXAMPLE 5 Leaves of a low-grade bright tobacco (X-1 2) were treated asdescribed in Example 1 with each of three lots of peptidase (Lots 2121,2574, and 3154 from Nutritional Biochemicals Corporation). Each lot wasused at a 0.3, 1.0, and 5.0% level of concentration.

Cigarettes made from the treated tobacco were compared with cigarettesprepared from unaged and forceaged leaves of the same low-grade stock.The forceaged leaves had been submitted to accelerated aging in a mannersimilar to that described in Industrial and Engineering Chemistry, 44,p. 284 (1952) by F. Darkes et al. Subjective evaluation of the smoke bya 39-member smoking panel showed that the cigarettes from theenzymetreated tobacco used at a 1% level were much preferred over thosefrom either the unaged or force-aged tobacco. The cigarettes made fromthe tobacco treated with 0.3 and 5.0% concentrations showed nostatistically significant improvement over the controls.

The data which illustrates the improvement in the cigar- .ettes treatedat the 1% level of enzyme concentration are given below. They areexpressed as t-test values. These values represent a statisticalevaluation based on precise laws of probabily. Because of variations insubjective evaluation, two samples may seem to be different even thoughthey have come from an identical source. The t-test determines thelimits within which this variation would fall with known probability.The magnitude of this difference indicates the probability that thesources are or are not identical, and that the differences are truedifferences. Because of the size of the sampling, these particularvalues can be interpreted to mean that the treated cigarettes werepreferred over the untreated controls at a 1001 probability level.

Table III Enzyme treated vs. unaged Enzyme treated vs. force-aged EnzymeLot N o. Harshness Taste Preier- Harshness Taste Prefer- Intensity enceIntensity ence t=3.28 15:2.48- t=2.3 No Sig t=2.65 t=2.2 t=2.18 No sigt=2.32 t=2.15 N0 sig- No slg t=3.2 t=2.3 N0 sig- No sig t=2.3 t=2.1.

We claim:

served. In a second test with 25 panel members, the preference for thecigarette from the treated tobacco over that from naturally-aged tobaccowas statistically significant. The evaluation also showed a trend towardbetter flavor and less harshness for the experimental cigarette.

Subjective evaluation of the cigarette from the enzymetreated tobaccoversus the cigarette from unaged, untreated tobacco showed that theexperimental cigarette was significantly less harsh and significantlypreferred. Fiftyseven panel members participated in the evaluation. Twoadditional evaluations of the same cigarettes by a 29- member and by a50-member panel showed a trend toward less harshness in the experimentalcigarette.

EXAMPLE 4 Old Belt tobacco (078X) was treated as described above 1. Aprocess for the treatment of tobacco, which comprises adding to thetobacco a non-living composition comprising a peptidic enzyme selectedfrom the group consisting of monopolypeptidases, dipolypeptidases, tri-.

polypeptidases, carboxypeptidase, endopeptidases, aminoacylases,aminopeptidases, dehydropeptidases and dipeptidases which havespecificity for amino acids, amino acid amides, dipeptides, tripeptides,polypeptides and dehydropeptides, said composition being added to thetobacco in an amount corresponding to from about 0.02 to about 0.15 partby weight of peptidic enzyme per parts of tobacco, said compositoncontaining from 98.5 to 99.5 parts by weight of water and from 0.5 to1.5 parts by weight peptidic enzyme, subjecting the resulting mixturewithout removing any portion thereof to a temperature of from 60 to F.,for a period of one to 200 hours, bringing the moisture content of saidmixture to a value of 9 to 15% by subjecting the mixture to atemperature of 60 to 80 F. and thereafter incorporating the entiretobaccowater-enzyme composition in a smoking product, whereby theresulting smoking product contains all of the products of the reactionsof the tobacco-water-enzyme composition and all of the unreactedcomponents of the tobacco-water-enzyme composition.

2. A process for the treatment of tobacco, which comprises adding to thetobacco a non-living composition comprising a peptidic enzyme selectedfrom the group consisting of monopolypeptidases, dipolypeptidases,tripolypeptidases, carboXypeptid-ases, endopeptidases, aminoacyla es,aminopeptidases, dehydropeptidases and dipeptidases, and a proteolyticenzyme selected from the group consisting of ficin, pepsin, trypsin,chymotrypsin, erepsin, fungal protease, protease and papain, saidcomposition being added to the tobacco in an amount corresponding tofrom about 0.02 to about 0.15 part by Weight of peptidic enzyme per 100parts of tobacco, said composition containing from 98.5 to 99.5 parts byweight of water and from 0.5 to 1.5 parts by Weight of peptidic andproteolytic enzyme, said peptidic and proteolytic enzyme being in theweight ratio of from 1:10 to 1:1 respectively, subject the resultingmixture, without removing any portion thereof to a temperature of from60 to 140 F., for a period of from one to 200 hours, bringing themoisture content of said mixture to a value of 9 to 15% by subjectingthe composition to a temperature of from 60 to 80 F., and thereafterincorporating the entire tobacco-water-enzyme composition in a smokingproduct, whereby the resulting smoking product contains all of theproducts of the reactions of the tobacco-water-enzyme composition andall of the unreacted components of the tobacco-water-enzyme composition.

References Cited by the Examiner UNITED STATES PATENTS 2,475,568 7/1949Moore 13-1-140 3,106,209 10 /1963 Torigian 131 3,132,651 5/1964 Kiefer131-141 FOREIGN PATENTS 252,121 10/1912 Germany.

OTHER REFERENCES Chemistry and Methods of Enzymes, by J. B. Summer andG. F. Somers, p. 172, published 1953 by Academic Press Inc., New York,NY.

Chemistry and Methods of Enzymes, by I. B. Summer and F. Somers, 3rdEdition, published 1953 by Academic Press Inc., New York, N.Y., pp. 166and 167.

Enzymes, by M. Dixon and E. C. Webb, pp. 266 and 267, published 1958 byAcademic Press Inc., New York, NY.

Shmuk: The Chemistry and Technology of Tobacco volume III, pp. 474 and475, published for National Science Foundation, Wash, DC. by IsraelProgram for Scientific Translations in 1961.

Websters Third New International Dictionary, p. 1674, published 1961 byG. and C. Merriam Co., Springfield, Mass.

SAMUEL KOREN, Primary Examiner.

1. A PROCESS FRO THE TREATMENT OF TOBACCO, WHICH COMPRISES ADDING TO THETOBACCO A NON-LIVING COMPOSITION COMPRISING A PEPTIDIC ENZYNE SELECTEDFROM THE GROUP CONSISTING OF MONOPOLYPEPTIDASES, DIPOLYPEPTIDASES,TRIPOLYPEPTIDASES, CARBOXYPEPTIDASE, ENDOPEPTIDASES, AMINOACYLASES,AMINOPEPTIDASES, DEHYDROPEPTIDASES AND DIPEPTIDASES WHICH HAVESPECIFICITY FOR AMINO ACIDS, AMINO ACID AMIDES, DIPEPTIDES, TRIPEPTIDES,POLYPEPTIDES AND DEHYDROPEPTIDES, SAID COMPOSITION BEING ADDED TO THETOBACCO IN AN AMOUNT CORRESPONDING TO FROM ABOUT 0.02 TO ABOUT 0.15 PARTBY WEIGHT OF PEPTIDIC ENZYNE PER 100 PARTS OF TOBACCO, SAID COMPOSITIONCONTAINING FROM 98.5 TO 99.5 PARTS BY WEIGHT OF WATER AND FROM 0.5 TO1.5 PARTS BY WEIGHT PEPTIDIC ENZYNE, SUBJECTING THE RESULTING MIXTUREWITHOUT REMOVING ANY PORTION THEREOF TO A TEMPERATURE OF FROM 60 TO140*F., FOR A PERIOD OF ONE TO 200 HOURS, BRINGING THE MOISTURE CONTENTOF SAID MIXTURE TO A VALUE OF 9 TO 15% BY SUBJECTING THE MIXTURE TO ATEMPERATURE OF 60 TO 80*F., AND THEREAFTER INCORPORATING THE ENTIRETOBACCOWATER-ENZYME COMPOSITION IN A SMOKING PRODUCT, WHEREBY THERESULTING SMOKING PRODUCT CONTAINS ALL OF THE PRODUCTS OF THE REACTIONSOF THE TOBACCO-WATER-ENZYME COMPOSITION AND ALL OF THE UNREACTEDCOMPONENTS OF THE TOBACCO-WATER-ENZYME COMPOSITION.